A. Iannuzzi

Sex chromosome abnormalities and sterility in river buffalo

Thirteen male river buffaloes, 119 females with reproductive problems (which had reached reproductive age but had failed to become pregnant in the presence of bulls) and two male co-twins underwent both clinical and cytogenetic investigation. Clinical analyses performed by veterinary practitioners revealed normal body conformation and external genitalia for most females. However, some subjects showed some slight male traits such as large base horn circumference, prominent withers and tight pelvis. Rectal palpation revealed damage to internal sex adducts varying between atrophy of Mullerian ducts to complete lack of internal sex adducts (with closed vagina). All bulls had normal karyotypes at high resolution banding, while 25 animals (23 females and 2 male co-twins) (20.7%) with reproductive problems were found to carry the following sex chromosome abnormalities: X monosomy (2 females); X trisomy (1 female); sex reversal syndrome (2 females); and free-martinism (18 females and 2 males). All female carriers were sterile.

Clinical, cytogenetic and molecular evaluation in a dog with bilateral cryptorchidism and hypospadias

The aim of this study was to estimate prognostic factors in a Dalmatian dog with bilateral cryptorchidism and hypospadias. Cytogenetic and molecular analyses revealed a normal karyotype (2n = 78,XY) and the presence of SRY, INSL3 and RXFP2 genes with a normal DNA sequence for SRY and RXFP2, while the INSL3 sequence differed slightly from the normal one due to a heterozygous nucleotide change involving amino acid 22 of the INSL3 dog precursor protein. Levels of plasmatic testosterone were only 0.01 ng/ml, while FSH and LH serum levels were not detectable. After the human chorionic gonadotropin (hCG) test, the serum testosterone level was 0.01 ng/ml. Therefore, the phenotypic aetiology of this subject can not be well-defined because cryptorchidism and hypospadias were frequent clinical features with high genetic heterogeneity.

Clinical, cytogenetic and molecular studies on sterile stallion and mare affected by XXY and sex reversal syndromes, respectively

Abstract Clinical, cytogenetic and molecular observations on a sterile stallion 2n = 65, XXY and a sterile mare 2n = 64, XY are reported. The XXY stallion was a pure case since all cells showed the same chromosome constitution. In the cells of mare XY, no SRY gene was found by both FISH- and molecular analyses. Both carriers show normal body conformation but were sterile because the stallion had no spermatozoa in the ejaculate, as revealed by microscope observation, and the mare showed the typical gonadal dysgenesis since both the uterus and ovaries were hypoplasic, as revealed by both rectal palpation and ultrasonic analysis. Although a mutation and / or deletion of SRY gene seems to be involved in the sex reversal, this syndrome is not yet fully understood. The possibility of other genes playing an important role in this syndrome and changes in the protein encoded by SRY are discussed.

Similar rates of chromosomal aberrant secondary oocytes in two indigenous cattle (Bos taurus) breeds as determined by dual-color FISH

In vitro-matured metaphase II (MII) oocytes with corresponding first polar bodies (I pb) from two indigenous cattle (Bos taurus) breeds have been investigated to provide specific data upon the incidence of aneuploidy. A total of 165 and 140 in vitro-matured MII oocytes of the Podolian (PO) and Maremmana (MA) breeds, respectively, were analyzed by fluorescence in situ hybridization using Xcen and five chromosome-specific painting probes. Oocytes with unreduced chromosome number were 13.3% and 6.4% in the two breeds, respectively, averaging 10.2%. In the PO, out of 100 MII oocytes + I pb analyzed, two oocytes were nullisomic for chromosome 5 (2.0%) and one disomic for the same chromosome (1.0%). In the MA, out of 100 MII oocytes + I pb, one oocyte was found nullisomic for chromosome 5 (1.0%) and one was disomic for the X chromosome (1.0%). Out of 200 MII oocytes + I pb, the mean rate of aneuploidy (nullisomy + disomy) for the two chromosomes scored was 2.5%, of which 1.5% was due to nullisomy and 1.0% due to disomy. By averaging these data with those previously reported on dairy cattle, the overall incidence of aneuploidy in cattle, as a species, was 2.25%, of which 1.25% was due to nullisomy and 1.0% due to disomy. The results so far achieved indicate similar rates of aneuploidy among the four cattle breeds investigated. Interspecific comparison between cattle (Xcen-5 probes) and pig (Sus scrofa domestica) (1-10 probes) also reveal similar rates. Further studies are needed that use more probes to investigate the interchromosomal effect. Establishing a baseline level of aneuploidy for each species/breed could also be useful for improving the in vitro production of embryos destined to the embryo transfer industry as well as for monitoring future trends of the reproductive health of domestic animals in relation to management errors and/or environmental hazards.

Numerical Sex Chromosome Aberrations and Abnormal Sex Development in Horse and Sheep

Gonadal dysgenesis and heterosexual conditions are often associated with sex chromosome abnormalities. In this study we report on 2 cases of abnormal sex development involving numerical sex chromosome aberrations in both horse and sheep. A 17-month-old Standardbred filly was sent to an equine fertility centre as an embryo donor due to its reduced size, being much smaller than a racehorse filly of the same age, which excluded it from an athletic career. External genitalia were clinically normal but manual palpation of the reproductive tract showed the presence of a small underdeveloped uterus and ovaries, as confirmed by ultrasonographic examination. Cytogenetic investigation by CBA-banding revealed an abnormal karyotype with X chromosome monosomy (2n = 63,X). A 18-month-old ewe showed distinct heterosexual traits with presence of a vulva (with enlarged clitoris), well-developed abdominal testes and mammary glands. Internal sex adducts were atrophic as seen after mating. Cytogenetic analysis revealed the presence of XX/XY mosaicism.

Sequential Cross-Species Chromosome Painting among River Buffalo, Cattle, Sheep and Goat: A Useful Tool for Chromosome Abnormalities Diagnosis within the Family Bovidae

The main goal of this study was to develop a comparative multi-colour Zoo-FISH on domestic ruminants metaphases using a combination of whole chromosome and sub-chromosomal painting probes obtained from the river buffalo species (Bubalus bubalis, 2n = 50,XY). A total of 13 DNA probes were obtained through chromosome microdissection and DOP-PCR amplification, labelled with two fluorochromes and sequentially hybridized on river buffalo, cattle (Bos taurus, 2n = 60,XY), sheep (Ovis aries, 2n = 54,XY) and goat (Capra hircus, 2n = 60,XY) metaphases. The same set of paintings were then hybridized on bovine secondary oocytes to test their potential use for aneuploidy detection during in vitro maturation. FISH showed excellent specificity on metaphases and interphase nuclei of all the investigated species. Eight pairs of chromosomes were simultaneously identified in buffalo, whereas the same set of probes covered 13 out 30 chromosome pairs in the bovine and goat karyotypes and 40% of the sheep karyotype (11 out of 27 chromosome pairs). This result allowed development of the first comparative M-FISH karyotype within the domestic ruminants. The molecular resolution of complex karyotypes by FISH is particularly useful for the small chromosomes, whose similarity in the banding patterns makes their identification very difficult. The M-FISH karyotype also represents a practical tool for structural and numerical chromosome abnormalities diagnosis. In this regard, the successful hybridization on bovine secondary oocytes confirmed the potential use of this set of probes for the simultaneous identification on the same germ cell of 12 chromosome aneuploidies. This is a fundamental result for monitoring the reproductive health of the domestic animals in relation to management errors and/or environmental hazards.

Analysis of chromosome damage by sister chromatid exchange (SCE) and redox homeostasis characterization on sheep flocks from Sardinian pasturelands.

Over the last decades, an increase of pollutants of diverse origin (industrial, military, mining, etc.) was recorded in several areas of Sardinia Island. We report the results of a multidisciplinary and complementary study based on cytogenetic and physiological analyses. The data obtained show the effects of the environmental impact on six sheep flocks (Sardinian breed) grazing on natural pasturelands next to possible polluted areas and compared to three herds grazing in different areas far from those potentially contaminated and used as control. Sister chromatid exchange (SCE) test was used as cytogenetic test to analyze chromosomal damages and it was performed on peripheral blood samples collected from 129 adult sheep (age > 4 years) randomly selected from polluted (92 animals) and control (37 animals) areas. Two types of cell cultures were performed: without (normal cultures) and with the addition of 5-BrdU. SCE-mean values estimated over 35 cells counted for each animal were 8.65 ± 3.40, 8.10 ± 3.50, 8.05 ± 3.08, 7.42 ± 3.34, 9.28 ± 3.56 and 8.38 ± 3.29 in the exposed areas, whereas the average values were 7.86 ± 3.31 in the control group. Significant increases (P < 0.01) of SCEs were found in three investigated areas of Southern Sardinia. Furthermore, sheep of the same flocks were characterized for blood redox homeostasis in order to define the potential targets of oxidative damage and to identify biomarkers of the extent of animal exposure to environmental contaminants. The plasma levels of Asc, Toc and Ret were found to be significantly lower (P < 0.001) in exposed sheep (I, II, IV and V) than in the control group. TAC as well as GPx and SOD activities were higher in control than in the exposed groups (P < 0.001). Finally, plasma levels of N-Tyr, PC, and LPO were significantly lower (P < 0.001) in the control group than in the exposed groups.

Frequency and distribution of rob(1;29) in eight Portuguese cattle breeds.

Cytogenetic investigations performed in eight Portuguese cattle breeds revealed the presence of rob(1;29) in both heterozygous and homozygous conditions in all, and five breeds, respectively, with variable percentages of carriers as follows: 41.0% in Arouquesa, 69.9% in Barrosa, 39.4% in Maronesa, 2.8% in Mirandesa, 8.5% in Marinhoa, 1.8% in Mertolenga, 21.3% in Raca Brava and 21.5% in Alentejana. CBA- and RBA-banding were performed to ascertain the chromosomes involved in the chromosome abnormality. A total of 1,626 animals were investigated. Reproductive parameters (number of calves per 100 cows) were higher in Mirandesa (80%) when compared with both Maronesa (75%) and Barrosa (70%) breeds, underlining that rob(1;29) reduces fertility in the carriers.

43rd Biennial American Cytogenetics Conference. May 4-7, 2014. The Omni Grove Park Inn. Asheville, North Carolina, USA: Abstracts

Abbott Molecular Affymetrix, Inc. Agilent Technologies Applied Spectral Imaging, Inc. BioDiscovery, Inc. BioDot, Inc. Cartagenia Cytocell/Rainbow Scientific, Inc. Genial Genetics/Rainbow Scientific, Inc. Illumina, Inc. International Collaboration for Clinical Genomics (ICCG) Leica Biosystems – CytoVision Leica Biosystems – Kreatech MetaSystems Miltenyi Biotec Oxford Gene Technology, Inc. SCC Soft Computer SciGene Transgenomic, Inc.

Molecular Characterization of Xp Chromosome Deletion in a Fertile Cow

A young cow of the Marchigiana breed (central Italy) with normal body conformation and external genitalia underwent routine cytogenetic analyses prior to its use for reproduction. After normal chromosome staining, only one X chromosome was observed with a normal diploid number (2n = 60) in all 200 studied cells. Subsequent cytogenetic analyses by using both CBA- and RBA-banding techniques evidenced that almost all the p arms of the other X chromosome was lacking. Detailed FISH-mapping analyses with BAC covering this Xp arm region demonstrated that this large chromosome region was deleted. RBA-banding showed that the deleted X was late replicating. CGH array analysis evidenced that deletion involves the Xp arm from the telomere to around 39.5 Mb, referring to the BosTau6 cattle genome assembly. This abnormality deletes about 40 Mb of the X chromosome sequence, but, despite the large number of genes deleted, none of them are programmed to escape from inactivation. This can explain the normal phenotype of the female which is actually pregnant. Finally, we evidenced, by analysis of an SNP mapped to the deleted region (SNP rs29024121), that the only normal (e.g. nondeleted) X chromosome present derives from the father. Hence, the deletion has a maternal origin.